DNA-protein fusions and uses thereof

ABSTRACT

Disclosed herein are molecules that include a deoxyribonucleic acid (DNA) covalently bonded to a protein and uses thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of co-pendingapplication, U.S. Ser. No. 60/110,549, filed Dec. 2, 1998.

BACKGROUND OF THE INVENTION

In general, the invention features DNA-protein fusions and their uses,particularly for the selection of desired proteins and theircorresponding nucleic acid sequences.

Recently, a combinatorial method was developed for the isolation ofproteins with desired properties from large pools of proteins (Szostaket al., U.S. Ser. No. 09/007,005; Szostak et al., WO98/31700; Roberts &Szostak, Proc. Natl. Acad. Sci. USA (1997) vol. 94, p. 12297-12302). Bythis method, the protein portion is linked to its encoding RNA by acovalent chemical bond. Due to the covalent nature of this linkage,selection experiments are not limited to the extremely mild reactionconditions that must be used for approaches that involve non-covalentcomplex formation such as ribosome display (Hanes & Plückthun, Proc.Natl. Acad. Sci. USA (1997) vol. 94, p. 4937-4942; He & Taussig, Nucl.Acids Res. (1997) vol. 25, p 5132-5143). However, precautions do need tobe taken during the selection process to minimize RNA degradation, sincethe accidental cleavage of ribo-bonds can result in the irreversibleloss of encoded information. For this reason, these selection proceduresare typically carried out using reaction media and equipment that arefree of ribonucleases or other deleterious contaminants.

SUMMARY OF THE INVENTION

The present invention provides methods for covalently tagging proteinswith their encoding DNA sequences. These DNA-protein fusions, which maybe used in molecular evolution and recognition techniques, arechemically more stable than RNA-protein fusions and therefore provide anumber of advantages (as discussed in more detail below).

Accordingly, in general, the invention features methods for generatingDNA-protein fusions. A first method involves: (a) linking a nucleic acidprimer to an RNA molecule (preferably, at or near the RNA 3′ end) , theprimer being bound to a peptide acceptor (for example, puromycin); (b)translating the RNA to produce a protein product, the protein productbeing covalently bound to the primer; and (c) reverse transcribing theRNA to produce a DNA-protein fusion.

A second method involves: (a) generating an RNA-protein fusion; (b)hybridizing a nucleic acid primer to the fusion (preferably, at or nearthe RNA 3′ end); and (c) reverse transcribing the RNA to produce aDNA-protein fusion.

In a preferred embodiment of the above methods, the method may furtherinvolve treating the product of step (c) to remove the RNA (for example,by contacting the product of step (c) with RNase H under conditionssufficient to digest the RNA). In additional preferred embodiments, thenucleic acid primer is a DNA primer; the translating step is carried outin vitro; and the nucleic acid primer has a hairpin structure. Inaddition, the primer may further include a photocrosslinking agent, suchas psoralen, and the primer may be crosslinked to an oligonucleotidewhich is bound to a peptide acceptor or, alternatively, may behybridized to the RNA molecule, followed by a linking step that iscarried out by photocrosslinking.

In related aspects, the invention also features a molecule including aDNA covalently bonded to a protein (preferably, of at least 10 aminoacids) through a peptide acceptor (for example, puromycin), as well as amolecule including a DNA covalently bonded to a protein, in which theprotein includes at least 10 amino acids.

In preferred embodiments of both of these aspects, the protein includesat least 30 amino acids, more preferably, at least 100 amino acids, andmay even include at least 200 or 250 amino acids. In other preferredembodiments, the protein is encoded by the DNA and is preferablyentirely encoded by the DNA; the molecule further includes a ribonucleicacid covalently bonded to the DNA; the protein is encoded by theribonucleic acid; and the DNA is double stranded.

In another related aspect, the invention features a population of atleast 10⁵, and preferably, at least 10¹⁴, DNA-protein fusions of theinvention, each fusion including a DNA covalently bonded to a protein.

In addition, the invention features selection methods which utilize theDNA-protein fusions described herein. A first selection method involvesthe steps of: (a) providing a population of DNA-protein fusions, eachincluding a DNA covalently bonded to a candidate protein; and (b)selecting a desired DNA-protein fusion, thereby selecting the desiredprotein or DNA.

A second selection method involves the steps of: (a) producing apopulation of candidate DNA-protein fusions, each including a DNAcovalently bonded to a candidate protein and having a candidate proteincoding sequence which differs from a reference protein coding sequence;and (b) selecting a DNA-protein fusion having an altered function,thereby selecting the protein having the altered function or itsencoding DNA.

In preferred embodiments, the selection step involves either binding ofthe desired protein to an immobilized binding partner or assaying for afunctional activity of the desired protein. In addition, the method mayfurther involve repeating steps (a) and (b).

In a final aspect, the invention features a solid support including anarray of immobilized molecules, each including a covalently-bondedDNA-protein fusion of the invention. In a preferred embodiment, thesolid support is a microchip.

As used herein, by a “population” is meant 10⁵ or more molecules (forexample, DNA-protein fusion molecules). Because the methods of theinvention facilitate selections which begin, if desired, with largenumbers of candidate molecules, a “population” according to theinvention preferably means more than 10⁷ molecules, more preferably,more than 10⁹, 10¹³, or 10¹⁴ molecules, and, most preferably, more than10¹⁵ molecules.

By “selecting” is meant substantially partitioning a molecule from othermolecules in a population. As used herein, a “selecting” step providesat least a 2-fold, preferably, a 30-fold, more preferably, a 100-fold,and, most preferably, a 1000-fold enrichment of a desired moleculerelative to undesired molecules in a population following the selectionstep. A selection step may be repeated any number of times, anddifferent types of selection steps may be combined in a given approach.

By a “protein” is meant any two or more naturally occurring or modifiedamino acids joined by one or more peptide bonds. “Protein” and “peptide”are used interchangeably herein.

By “RNA” is meant a sequence of two or more covalently bonded, naturallyoccurring or modified ribonucleotides. One example of a modified RNAincluded within this term is phosphorothioate RNA.

By “DNA” is meant a sequence of two or more covalently bonded, naturallyoccurring or modified deoxyribonucleotides.

By a “nucleic acid” is meant any two or more covalently bondednucleotides or nucleotide analogs or derivatives. As used herein, thisterm includes, without limitation, DNA, RNA, and PNA.

By a “peptide acceptor” is meant any molecule capable of being added tothe C-terminus of a growing protein chain by the catalytic activity ofthe ribosomal peptidyl transferase function. Typically, such moleculescontain (i) a nucleotide or nucleotide-like moiety (for example,adenosine or an adenosine analog (di-methylation at the N-6 aminoposition is acceptable)), (ii) an amino acid or amino acid-like moiety(for example, any of the 20 D- or L-amino acids or any amino acid analogthereof (for example, O-methyl tyrosine or any of the analogs describedby Ellman et al., Meth. Enzymol. 202:301, 1991), and (iii) a linkagebetween the two (for example, an ester, amide, or ketone linkage at the3′ position or, less preferably, the 2′ position); preferably, thislinkage does not significantly perturb the pucker of the ring from thenatural ribonucleotide conformation. Peptide acceptors may also possessa nucleophile, which may be, without limitation, an amino group, ahydroxyl group, or a sulfhydryl group. In addition, peptide acceptorsmay be composed of nucleotide mimetics, amino acid mimetics, or mimeticsof the combined nucleotide-amino acid structure.

By an “altered function” is meant any qualitative or quantitative changein the function of a molecule.

By “binding partner,” as used herein, is meant any molecule which has aspecific, covalent or non-covalent affinity for a portion of a desiredDNA-protein fusion. Examples of binding partners include, withoutlimitation, members of antigen/antibody pairs, protein/inhibitor pairs,receptor/ligand pairs (for example cell surface receptor/ligand pairs,such as hormone receptor/peptide hormone pairs), enzyme/substrate pairs(for example, kinase/substrate pairs), lectin/carbohydrate pairs,oligomeric or heterooligomeric protein aggregates, DNA bindingprotein/DNA binding site pairs, RNA/protein pairs, and nucleic acidduplexes, heteroduplexes, or ligated strands, as well as any moleculewhich is capable of forming one or more covalent or non-covalent bonds(for example, disulfide bonds) with any portion of a DNA-protein fusion.

By a “solid support” is meant, without limitation, any column (or columnmaterial), bead, test tube, microtiter dish, solid particle (forexample, agarose or sepharose), microchip (for example, silicon,silicon-glass, or gold chip), or membrane (for example, the membrane ofa liposome or vesicle) to which an affinity complex may be bound, eitherdirectly or indirectly (for example, through other binding partnerintermediates such as other antibodies or Protein A), or in which anaffinity complex may be embedded (for example, through a receptor orchannel).

The present invention provides methods for the creation of fusionsbetween proteins and their encoding cDNAs. These constructs possessgreatly enhanced chemical stability, first, due to the DNA component ofthe fusion and, second, due to the covalent bond linking of the DNA andprotein moieties. These properties allow for easier handling of thefusion products and thereby allow selection and recognition experimentsto be carried out under a range of reaction conditions. In addition, thepresent invention facilitates applications where a single-strandednucleic acid portion is mandatory, for example, in hybridization assaysin which the coding fusions are immobilized to a solid support. Inaddition, incubations may be performed under more rigorous conditions,involving high pH, elevated concentrations of multivalent metal ions,prolonged heat treatment, and exposure to various biological materials.Finally, single-stranded DNA is relatively resistant to secondarystructure formation, providing a great advantage for techniquesinvolving or requiring nucleic acid hybridization steps.

In addition, the methods of the present invention allow for theproduction of fusions involving DNA and protein components of anylength, as well as fusion libraries of high complexity.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a method for the generation ofDNA-Protein fusions (Type A1) that involves ligation of apuromycin-modified DNA hairpin-like structure to an mRNA molecule.

FIG. 2 is a schematic illustration of a method for the generation ofbranched hairpin structures.

FIG. 3 is a schematic illustration of a method for the synthesis ofpuromycin-5′-phosphoramidite.

FIG. 4 is a schematic illustration of a method for the generation ofbranched hairpin structures.

FIG. 5 is a schematic illustration of a method for the generation ofDNA-protein fusions that involves photocrosslinking of a5′-psoralen-modified primer DNA to a suitable linker that bears a3′-puromycin.

FIG. 6 is a schematic illustration of exemplary methods for the chemicalligation of mRNA and DNA molecules.

FIG. 7 is a schematic illustration of a method for the synthesis ofhydrazide phosphoramidite.

FIG. 8 is a schematic illustration of a method for the synthesis ofhydrazine phosphoramidite.

FIG. 9 is a schematic illustration of a method for the generation ofDNA-protein fusions that involves chemical crosslinking of apuromycin-modified linker to the 3′-end of an mRNA molecule.

FIG. 10 is a schematic illustration of a method for the generation ofDNA-protein fusions that involves psoralen-mediated photocrosslinking ofa combined linker/reverse transcription primer construct to the 3′-endof an mRNA molecule.

FIG. 11 is a schematic illustration of an alternative method for thegeneration of DNA-protein fusions that involves psoralenphotocrosslinking of a combined linker/reverse transcription primerconstruct.

FIG. 12 is a schematic illustration of a method for the generation ofDNA-protein ions that involves crosslinking of a reverse transcriptionprimer to a preexisting mRNA-linker construct.

FIG. 13 is a schematic illustration of a method for the generation ofDNA-protein fusions that involves crosslinking of a reversetranscription primer to a preexisting mRNA-protein fusion.

FIG. 14 is a schematic illustration of the oligonucleotide constructs(SEQ ID NOS: 1-6) used for the preparation of the exemplary DNA-proteinfusions described herein.

FIG. 15 is a schematic illustration of the preparation of Type C2DNA-proteins fusions.

FIG. 16 is a photograph illustrating a product analysis of the Type C2DNA-protein fusions.

FIG. 17 is a schematic illustration of the preparation of Type B3DNA-protein fusions.

FIG. 18 is a schematic illustration of the preparation of Type B2DNA-protein fusions.

FIG. 19 is a photograph illustrating the resistance analysis of Type B3DNA-protein fusions against nuclease and base treatment.

FIG. 20 is a graph illustrating the experimentally determined half-livesof RNA- and DNA-protein fusion products in the presence of cell membranepreparations.

DETAILED DESCRIPTION

There are now provided below a number of exemplary techniques for theproduction of DNA-protein fusions, and descriptions for their use. Theseexamples are provided for the purpose of illustrating, and not limiting,the invention.

Type A1

Template-Directed Ligation of a Puromycin-Modified Hairpin-LikeStructure to an mRNA

According to a first exemplary approach, DNA-protein fusions aregenerated by ligating a puromycin-modified DNA hairpin-like structure toan mRNA molecule, as illustrated in FIG. 1. The first step of thisprocedure is the attachment of puromycin to the hairpin, and this may beaccomplished by a number of techniques, one of which is shown in FIG. 2.By this approach, a DNA hairpin is synthesized with apuromycin-terminated side chain branching out from the DNA molecule.This construct may be generated using an asymmetric branchedphosphoramidite (Clontech, Palo Alto, Calif.) in any standard automatedDNA synthesis of the hairpin structure (see, for example, User Guide toExpedite Nucleic Acid Synthesis System, Perseptive Biosystems,Framingham, Mass.), followed by the addition of a 5′-phosphate using achemical phosphorylation reagent (Glen Research, Sterling Va.).

Subsequently the protecting group is selectively removed from the branch(Product Protocol for Asymmetric Branching Phosphoramidite, Clontech,Palo Alto, Calif.), followed by the attachment of the linker portionthrough standard automated DNA synthesis. Before reaching the end of thelinker, the strand orientation is reversed by the addition of a few5′-phosphoramidites (Glen Research, Sterling, Va.). Finally, thesynthesis is terminated through attachment of thepuromycin-5′-phosphoramidite, preferably using the synthetic techniqueshown in FIG. 3. In FIG. 3, steps (a)-(c) may be carried out asdescribed in Greene & Wuts (Protective Groups in Organic Synthesis,2^(nd) ed. (1991) John Wiley & Sons, Inc., New York, N.Y.), and step (d)may be carried out as described in Beaucage (Methods in MolecularBiology, vol. 20, Protocols for Oligonucleotides and Analogs, ed. S.Agarwal (1993) Humana Press, Totowa, N.J., pp. 33-61).

Alternatively, the puromycin-modified branched hairpin may besynthesized as shown in FIG. 4. By this technique, synthesis isinitiated from a puromycin-CPG solid support (Glen Research, Sterling,Va.) by first synthesizing the linker portion, followed by incorporationof the branched amidite (Clontech, Palo Alto, Calif.) and addition ofthe 5′-portion of the hairpin. After deprotection of the branch, the3′-arm of the hairpin is added by using nucleoside-5′-phosphoramidites(Glen Research, Sterling, Va.).

By either of the above approaches, in the next step, the mRNA is ligatedto the hairpin, for example, using T4 DNA ligase and the 3′-overhang asa template (Sambrook, Fritsch & Maniatis Molecular Cloning (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Ribosomaltranslation of the RNA then leads to protein synthesis with subsequentfusion formation (see, for example, Szostak et al., U.S. Ser. No.09/007,005 and U.S. Ser. No. 09/247,190; Szostak et al., WO98/31700;Roberts & Szostak, Proc. Natl. Acad. Sci. USA (1997) vol. 94, p.12297-12302). In one particular embodiment, the branching point islocated in the loop region of the hairpin. Other positions of thebranching point (e.g., within the stem structure) may also be utilized.In addition, while a dA_(n) linker of between approximately 10-60nucleotides, and more preferably approximately 30 nucleotides, isutilized, both the length and the chemical composition (e.g., PEG (GlenResearch, Sterling, Va.) rather than dA_(n)) of the linker may beoptimized.

In a final step, the RNA portion of the construct is reverse transcribedinto cDNA (for example, as described in Sambrook, Fritsch & Maniatis,Molecular Cloning, (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.) using the hairpin 3′ end as a primer. Optionaldigestion of the mRNA by RNase H (see, for example, Sambrook, Fritsch &Maniatis Molecular Cloning, (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.) yields a single stranded DNA-protein fusion.

This method also facilitates the formation of truncated DNA transcriptsby adding didesoxynucleoside triphosphates during transcription (see,for example, Sanger, Science (1981) vol. 214, p. 1205-1210). Suchtruncated DNA-protein fusions are useful in protein display experiments(Kuimelis et al., U.S. Ser. No. 60/080,686, filed Apr. 3, 1998), forexample, where only the 3′-region of the original message (now the5′-region of the DNA transcript) is used for hybridization withimmobilized oligonucleotide probes.

Type A2

Crosslinking of a Puromycin-Modified Linker to a Primer DNA

As an alternative to the hairpin-like construct described above, aclosely related structure may also be prepared through photocrosslinkingof a 5′-psoralen-modified primer DNA with a suitable linker that bears a3′-puromycin. An exemplary crosslinking method is illustrated in FIG. 5.In this method, the puromycin-bearing linker may be constructed asdescribed, for example, in Szostak et al., U.S. Ser. No. 09/007,005 andU.S. Ser. No. 09/247,190; Szostak et al., WO98/31700; Roberts & Szostak,Proc. Natl. Acad. Sci. USA (1997) vol. 94, p. 12297-12302. Thepsoralen-modified primer may be generated and the photocrosslinking stepcarried out as described, for example, in Pieles & Englisch, Nucl. AcidsRes. (1989) vol. 17, p. 285-299. The remaining steps may be carried outas described above. This approach does not require the use ofnon-standard nucleoside/puromycin-5′-phosphoramidites (i.e., which wereused during the automated synthesis of the hairpin-linker structure),providing an advantage over the hairpin method. Again, as above, while adA_(n) linker of between approximately 10-60 nucleotides, and morepreferably approximately 30 nucleotides, is utilized, both the lengthand the chemical composition (e.g., PEG (Glen Research, Sterling, Va.)rather than dA_(n)) of the linker may be optimized.

In addition, for each of the Type A1 and Type A2 methods, the ligationreaction between the mRNA and the DNA portion of the construct may becarried out by several alternative techniques. For example, in additionto the enzymatic ligation with T4 DNA ligase described above, this stepmay be accomplished using chemical methods. In one particular example,the 5′-end of the hairpin may be modified with one (or multiple)amino-groups using the appropriate phosphoramidite (Clontech, Palo Alto,Calif.). After periodate oxidation of the 3′-end of the RNA, the twosubstrates may be joined through a reductive amination reaction. This isillustrated as scheme “A” in FIG. 6 and is described, for example, inLemaitre et al., Proc. Natl. Acad. Sci. USA (1987) vol. 84, p. 648-652.Alternatively, this chemical ligation step may involve carbohydrazide orhydrazine modified structures for hydrazone formation or reductiveamination. These approaches are illustrated in FIG. 6, respectively, asschemes “B” and “C” and are described, respectively, in Gosh et al.(Anal. Biochem. (1989) vol. 178, p. 43-51) and Proudnikov & Mirzabekov(Nucl. Acids Res. (1996) vol. 24 p. 4535-4542). Hydrazidephosphoramidite synthesis may be carried out as shown in FIG. 7, andhydrazine phosphoramidite synthesis as shown in FIG. 8 and as describedin Greene & Wuts (Protective Groups in Organic Synthesis, 2^(nd) ed.(1991) John Wiley & Sons, Inc., New York, N.Y. (steps (a) and (c)),Proudnikov & Mirzabekov (Nucl. Acids Res. (1996) vol. 24 p. 4535-4542(step b)), and Beaucage (Methods in Molecular Biology, vol. 20,Protocols for Oligonucleotides and Analogs, ed. S. Agarwal (1993) HumanaPress, Totowa, N.J., pp. 33-61 (step (e)).

Types B1-B3

Chemical Crosslinking to the 3′-end of an mRNA

Yet another approach to the generation of DNA-protein fusions involvesthe chemical crosslinking of a puromycin-modified linker to the 3′-endof an mRNA molecule. Such crosslinking may be accomplished by a numberof approaches.

One exemplary approach is shown schematically in FIG. 9. In thisapproach (“B1”), an oligonucleotide is synthesized that bears a reactivegroup (e.g., one of the amino derivatives, hydrazides, or hydrazinesdescribed above) located between the primer and the linker regions.Duplex formation of the RNA and the primer site takes place immediatelyadjacent to this reactive group, which then is allowed to react with theperiodate-oxidized 3′-end of the RNA leading to a crosslink (as shown inFIG. 6 and as described above). This reaction may occur throughreductive amination (FIG. 6, scheme “A” or “C”; Lemaitre et al., Proc.Natl. Acad. Sci. USA (1987) vol. 84, p. 648-652; Proudnikov &Mirzabekov, Nucl. Acids Res. (1996) vol. 24 p. 4535-4542) or hydrazoneformation (FIG. 6, scheme “B”; Gosh et al., Anal. Biochem. (1989) vol.178, p. 43-51). Following translation and fusion formation (Szostak etal., U.S. Ser. No. 09/007,005 and U.S. Ser. No. 09/247,190; Szostak etal., WO98/31700; Roberts & Szostak, Proc. Natl. Acad. Sci. USA (1997)vol. 94, p. 12297-12302), the primer is extended by reversetranscriptase on the RNA template and an optional RNase H digestion stepis carried out, generating the DNA-protein fusion (FIG. 9).

As in methods A1 and A2 above, the strand direction of the linkerportion's terminal nucleotides is reversed, which can be accomplished bythe use of 5′-phosphoramidites (Glen Research, Sterling, Va.) duringsynthesis.

In yet another exemplary crosslinking approach (“B2”), a photoreactivepsoralen moiety is included in the linker as a reactive group (FIG. 10).Such a construct may be synthesized using a psoralen-modifieddesoxynucleotide phosphoramidite (Pieles et al., Nucleic Acids Res.(1989) vol. 17, p. 8967-8978) or by incorporating a branchedphosphoramidite (Clontech, Palo Alto, Calif.) to which a standardpsoralen phosphoramidite is attached (Glen Research, Sterling, Va.).Following hybridization of the linker to the target RNA, crosslinkformation is achieved through irradiation with UV-light, for example, asdescribed in Pieles and Englisch (Nucl. Acids Res. (1989) vol. 17, p.285-299). The resulting construct is then subjected to translation andfusion formation (Szostak et al., U.S. Ser. No. 09/007,005 and U.S. Ser.No. 09/247,190; Szostak et al., WO98/31700; Roberts and Szostak, Proc.Natl. Acad. Sci. USA (1997) vol. 94, p. 12297-12302). Reversetranscription and RNase H digestion yields the final DNA-proteinfusions.

Alternatively, crosslinking may be accomplished using a combinedlinker/reverse transcriptase primer construct as depicted in FIG. 11(“B3”). In a variant of the above approach, the psoralen moiety is notdirectly attached between the linker and primer region, but ratherconnected to a short DNA branch. This DNA portion also hybridizes to thetarget RNA and thus provides an optimized double-stranded environmentfor the psoralen to react (Pieles and Englisch, Nucl. Acids. Res. (1989)vol. 17, p. 285-299). Preparation of DNA-protein fusions using thispsoralen construct may be carried out as described above.

Types C1 and C2

Crosslinking of the Reverse Transcription Primer to PreexistingmRNA-Linker Constructs

Another method for generating DNA-protein fusions is shown schematicallyin FIG. 12. By this approach, RNA is initially ligated to a linkermolecule as previously described (Szostak et al., U.S. Ser. No.09/007,005 and U.S. Ser. No. 09/247,190; Szostak et al., WO98/31700;Roberts & Szostak, Proc. Natl. Acad. Sci. USA (1997) vol. 94, p.12297-12302). In a subsequent step, a suitable primer bearing a5′-photocrosslinking reagent (e.g., psoralen, Glen Research, Sterling,Va.) is annealed to the RNA-linker product. Irradiation with lightfurnishes a covalent crosslink between the two oligonucleotide strands(as described, for example, in Pieles & Englisch, Nucl. Acids Res.(1989) vol. 17, p. 285-299). As in methods Type A1, A2, and B1-B3 above,translation and fusion formation may be carried out, followed by areverse transcription step and an optional RNase H digestion step toyield DNA-protein fusions (FIG. 12).

Alternatively, as shown in FIG. 13, the initial steps of the aboveprocedure may be carried out in the opposite order. This approach allowstranslation and fusion formation to be performed prior to crosslinkingand reverse transcription. Accordingly, this method allows for the useof previously described and well established reaction conditions andcomponents for translation and RNA-protein fusion formation.

Experimental Results

Exemplary techniques described above were carried out to demonstrateDNA-protein fusion formation. These experiments made use of theoligonucleotides depicted in FIG. 14.

Model RNA substrates 1: GGG ACA AUU ACU AUU UAC AAU UAC AAU GGA CUA CAAGGA CGA UGA CGA UAA GGG CGG CUG GUC CCA CCC CCA GUU CGA GAA GGC AUC CGCU (SEQ ID NO: 7); 2: GGG ACA AUU ACU AUU UAC AAU UAC AAU GGA CUA CAA GGACGA UGA CGA UAA GGG CGG CUG GUC CCA CCC CCA GUU CGA GAA GGC AUC CGC UCUUUC ACU AUA (SEQ ID NO: 8); and 3: GGG ACA AUU ACU AUU UAC AAU UAC AAUGGA CUA CAA GGA CGA UGA CGA UAA GGG CGG CUG GUC CCA CCC CCA GUU CGA GAAGGC AUC CGC UAU UUA AAA AAA AAA AAA AAA AAA A (SEQ ID NO: 9) weresynthesized by T7 transcription (Megashortscript transkiption kit,Ambion, Austin, Tex.) using appropriate dsDNA templates. Followingtranscription, the RNAs were purified by denaturing polyacrylamide gelelectrophoresis.

The modified oligonucleotides 4: 5′ pd(AAA AAA AAA ACG GCT ATA TAA AAAAAA CC)- Pu (SEQ ID NO: 10); 5: 5′ psoralen C2-TAG CCG TTT TTT TTT TAGCGG ATG C (SEQ ID NO: 11); 6: 5′ d(cgt agg cga gaa agtgat)-branch[psoralen C6]-d(AAA AAA AAA AAA AAA AAA AAA AAA AAA CC)-Pu(SEQ ID NO: 12); and 7: 5′ ggt caa gct ctt-branch[5′ psoralen C6-TAG CGGATG C 3′] spacer₆ CC-Pu (SEQ ID NO: 13) [[uppercase=standardDNA-3′-phosphoramidites; lowercase=DNA-5′-phosphoramidites;spacer=spacer-9 phosphoramidite; Pu=puromycin-CPG (all from GlenResearch, Sterling, Va.); branch=asymmetric branching amidite (Clontech,Palo Alto, Calif.)] were synthesized on an Expedite Synthesizer Model8909 (PerSeptive Biosystems, Framingham, Mass.) according to recommendedprotocols for the corresponding phosphoramidites. For the branchedconstructs 6 and 7, the main chain was synthesized first and concludedwith a final capping step. Next, the levulinyl protecting group wasremoved from the branching unit through treatment with 0.5 M hydrazinemonohydrate in pyridine-acetic acid for 15 minutes at room temperature.Automated synthesis was then resumed and the side chain sequences(indicated in square brackets) were attached. The oligos were fullydeprotected in concentrated ammonium hydroxide for 8 hours at 55° C. andpurified by denaturing gel electrophoresis.

The DNA sequences 8: d(TTT TTT TTT TAG CGG ATG C) (SEQ ID NO: 14) and 9:d(TAA TAC GAC TCA CTA TAG GGA CAA TTA CTA TTT ACA ATT) (SEQ ID NO: 15)were purchased from Oligos etc. (Wilsonville, Oreg.) and used withoutfurther purification.

Type C2 DNA-Protein Fusion Formation

Type C2 DNA-protein fusion formation was demonstrated as follows (FIG.15). RNA 1 and linker 4 were hybridized to template DNA 8 andenzymatically ligated by T4 DNA ligase as previously described (Szostaket al., U.S. Ser. No. 09/007,005 and U.S. Ser. No. 09/247,190; Szostaket al., WO98/31700; Roberts and Szostak, Proc. Natl. Acad. Sci. USA(1997) Vol. 94, p. 12297-12302). After purification by electrophoresison a denaturing polyacrylamide gel, the resulting mRNA-linker constructwas used as a template for in vitro translation using rabbitreticulocyte lysate kits from Ambion. Reaction mixtures contained 50pmole ligated mRNA 10, 10 mM creatine phosphate, 150 mM potassiumacetate, 0.5 mM magnesium chloride, 0.1 mM of each amino acid exeptmethionine, 150 μCi[³⁵S] methionine (Amersham, Arlington Heights, Ill.)and 67% v/v of lysate in a total volume of 300 μl and were carried outfor 30 minutes at 30° C. To promote the subsequent fusion formation, KCland MgCl₂ were added to 590 mM and 50 mM final concentrations,respectively, in a volume of 500 μl. Incubation was continued for 60minutes at 20° C. Products were isolated by diluting the lysate into 10ml of binding buffer (100 mM Tris pH 8.0, 10 mM EDTA, 1 M NaCl, 0.25%v/v Triton X-100) and adding 10 mg oligo-dT cellulose type 7 (Pharmacia,Piscataway, N.J.). Samples were rotated for 60 minutes at 4° C., and thesolid support was then washed with 5 ml ice-cold binding buffer that wasdevoid of EDTA, followed by elution with 100 μl aliquots of water.Fusion product was found in fractions 2 and 3, and these fractions werecombined. The total yield of fusion 11 was determined by scintillationcounting of the incorporated [³⁵S] methionine to be 1.6 pmole (3.2% ofinput RNA).

For the conversion of the RNA-protein fusions 11 into DNA-proteinfusions 13, the following reactions were performed (FIG. 15). First, 20μl of the above oligo-dT-purified material 11 was mixed with 0.5 μlprimers 5 (50 μM) and 6 μl first strand buffer (Superscript II kit fromGibcoBRL; 250 mM Tris-HCl pH 8.3, 375 KCl, 15 mM MgCl₂) and brieflyheated to 80° C. for 2 minutes, followed by slowly cooling to 0° C.Psoralen photocrosslink formation was induced by irradiating the samplefor 15 minutes at 0° C. with λ>310 nm [450 W medium pressure immersionlamp (ACE Glass, Vineland, N.J.) equipped with a Pyrex absorption sleevein a Quartz immersion well]. Next, 0.6 μl of a dNTP mix (25 mM each), 3μl of 0.1 M DTT, and 0.4 μl (80 units) Superscript II reversetranscriptase were added, and cDNA synthesis was carried out for 60minutes at 42° C. The RNA portion was then removed by continuingincubation for 60 minutes at 37° C. after addition of 0.5 μl (1 unit)RNase H (Promega, Madison, Wis.). Finally, double-stranded DNA 14 wasgenerated by adding 50 pmoles of primer 9 and incubating for another 60minutes at 42° C. Control reactions with non-crosslinked samples wereperformed as indicated in FIG. 15. Product analysis was performed byelectrophoresis on denaturing 6% TBE-Urea gels (Novex, San Diego,Calif.), followed by visualization of the [³⁵S]-labelled product bandsby exposure on a phosphorimager screen (FIG. 16).

Samples were applied to the gel in the same order as they appear in FIG.15, beginning with RNA-protein fusion 11 and following the reactionpathway with and without having been photocrosslinked. As indicated inFIG. 16, the gel mobilities correspond well with the expected behaviorand clearly confirm the constitution of DNA-protein fusion 13.

Type B3 DNA-Protein Fusion Formation

Type B3 DNA-protein fusion formation was demonstrated as follows (FIG.17). The branched linker-construct 7 (5 μM) was annealed to the targetRNA 3 (2.5 μM) in 25 mM Tris buffer pH 7.0 containing 100 mM NaCl andcrosslinked by irradiation for 15 minutes at room temperature in aborosilicate glass vial (Kimble/Kontes, Vineland, N.J.) using a handheldmultiwavelength UV lamp model UVGL-25 (UVP, Upland, Calif.) set to longwave. Product analysis was performed by electrophoresis on a 6% TBE-Ureapolyacrylamide gel followed by visualization by UV shadowing. Theseresults indicated nearly quantitative conversion of the startingmaterial (gel “A” in FIG. 17). The photoligated product RNA was used forin vitro translation without further separation from remaining unligatedRNA and excess linker. In vitro translation and fusion formationreactions were performed as described for Type C2 above, with 100 pmoleinput RNA in a 300 μl total volume. After purification on oligo-dTcellulose, 5.5 pmole RNA-fusion 15 was obtained. Its conversion intosingle-stranded and double-stranded DNA-protein fusions 16 and 17,respectively, was done by reverse transcription (Superscript II kit,GibcoBRL, Grand Island, N.Y.) and RNase H (Promega, Madison, Wis.)treatment as described for Type C2 fusions (gel “B” in FIG. 17).

Type B2 DNA-Protein Fusion Formation

Type B2 DNA-protein fusion formation was demonstrated as outlined inFIG. 18. Specifically, following the procedure outlined for Type B3fusions above, RNA 2 was crosslinked to linker 6. Following denaturingpolyacrylamide electrophoresis, the ligated product 18 was isolated in12% yield. In vitro translation, fusion formation, and preparation ofDNA-protein fusions 19 were carried out as described for Type B3 fusionsabove, with similar efficiencies of fusion formation.

DNA-Protein Fusion Stability Tests

To evaluate the nuclease and base resistance of DNA fusions incomparison with the corresponding RNA fusions, the following experimentswere carried out. To 10 μl DNA-fusion 16 (Type B3) or RNA-fusion 15 inreverse transcription buffer was added either 0.2 μl (0.4 units) RNaseH, 0.2 μl (2 units) RNase I, 0.2 μl (0.6 units) T4 DNA polymerase (3′-5′exonuclease activity), or 2.5 μl of 2.0 M NaOH. Samples were incubatedfor 30 minutes at 37° C. and then analyzed on a 4-12% NuPage gel (Novex,San Diego, Calif.) followed by autoradiography. Results are shown inFIG. 19 and confirm the increased stability of DNA fusions againstribonucleases and base treatment.

To test stability of DNA fusion constructs in biological media, 5 nM ofeither RNA fusions 11 or 12, or DNA fusions 13 or 14 (Type C2) wereincubated with 3 μg/μl CHO-K1 cell membranes (Receptor Biology,Beltsville, Md.) in 50 mM Tris-HCl pH 8.3, 75 mM KCl, 3 mM MgCl₂, and 10mM DTT at room temperature. Additional samples of RNA-fusions 11 and 12were prepared containing 20 mM vanadyl ribonucleside complex (“VRC”) toinhibit ribonuclease activity. Aliquots were taken after 0, 5, 15, 30,60, 120 minutes, and 24 hours and analyzed by electrophoresis on 4-12%NuPage gels (Novex) followed by exposure on a phosphorimager screen. Therelative amounts of remaining fusion were plotted against incubationtime and half-lives graphically extracted from the resulting curves. Asindicated in FIG. 20, all constructs showed more than 50% decay duringthe initial two hour period except for dsDNA fusion 14, which appearedto be entirely stable under the conditions tested. Following a 24 hourincubation, all fusion constructs were completely degraded due to eithernuclease or protease activity.

In Vitro Selection of Desired Proteins

The DNA-protein fusions described herein may be used in any selectionmethod for desired proteins, including molecular evolution andrecognition approaches. Exemplary selection methods are described, forexample, in Szostak et al., U.S. Ser. No. 09/007,005 and U.S. Ser. No.09/247,190; Szostak et al., WO98/31700; Roberts & Szostak, Proc. Natl.Acad. Sci. USA (1997) vol. 94, p. 12297-12302; Lipovsek et al., U.S.Ser. No. 60/096,818 and U.S. Ser. No. 09/374,962; and Kuimelis et al.U.S. Ser. No. 60/080,686 and U.S. Ser. No. 09/282,734, all herebyincorporated by reference.

Use

The DNA-protein fusions described herein may be used for any applicationpreviously described or envisioned for RNA-protein fusions. Commercialuses include the isolation of polypeptides with desired propertiesthrough in vitro evolution techniques (see, for example, Szostak et al.,U.S. Ser. No. 09/007,005 and U.S. Ser. No. 09/247,190; Szostak et al.,WO98/31700; Roberts & Szostak, Proc. Natl. Acad. Sci. USA (1997) vol.94, p. 12297-12302)), screening of cDNA libraries that are derived fromcellular mRNA (see, for example, Lipovsek et al., U.S. Ser. No.60/096,818, filed Aug. 17, 1998), and the cloning of new genes on thebasis of protein-protein interactions (Szostak et al., U.S. Ser. No.09/007,005 and U.S. Ser. No. 09,247,190; Szostak et al., WO98/31700), aswell as the use of these fusions in protein display experiments(Kuimelis et al. U.S. Ser. No. 60/080,686 and U.S. Ser. No. 09/282,734).In addition, the DNA-protein fusions described herein may be used inbinding and molecular recognition assays that involve biologicalmaterials that presumably contain ribonucleases, such as whole cells,lysates, or biological fluids. These DNA-protein fusions may be used forany appropriate therapeutic, diagnostic, or research purpose,particularly in the pharmaceutical and agricultural areas.

15 1 41 DNA Artificial Sequence misc_feature (1)...(41) n = A,U,C or G 1nnngcauccg cuaaaaaaaa aacggctata taaaaaaaac c 41 2 25 DNA ArtificialSequence DNA linker 2 cgtaggcgat tttttttttg ccgat 25 3 51 RNA ArtificialSequence misc_feature (1)...(34) n = A,U,C or G 3 nnnnnccagu ucgagaaggcauccgcuauu uaaaaaaaaa aaaaaaaaaa a 51 4 22 DNA Artificial Sequence DNAlinker 4 ggtcaagctc ttcgtaggcg at 22 5 23 RNA Artificial Sequencemisc_feature (1)...(23) n = A,U,C or G 5 nnngcauccg cucuuucacu aua 23 621 DNA Artificial Sequence DNA linker 6 cgtaggcgag aaagtgatac c 21 7 91RNA Artificial Sequence Model RNA substrate 7 gggacaauua cuauuuacaauuacaaugga cuacaaggac gaugacgaua agggcggcug 60 gucccacccc caguucgagaaggcauccgc u 91 8 102 RNA Artificial Sequence Model RNA substrate 8gggacaauua cuauuuacaa uuacaaugga cuacaaggac gaugacgaua agggcggcug 60gucccacccc caguucgaga aggcauccgc ucuuucacua ua 102 9 115 RNA ArtificialSequence Model RNA substrate 9 gggacaauua cuauuuacaa uuacaauggacuacaaggac gaugacgaua agggcggcug 60 gucccacccc caguucgaga aggcauccgcuauuuaaaaa aaaaaaaaaa aaaaa 115 10 29 DNA Artificial Sequence DNA linker10 aaaaaaaaaa cggctatata aaaaaaacc 29 11 25 DNA Artificial Sequence DNAlinker 11 tagccgtttt ttttttagcg gatgc 25 12 47 DNA Artificial SequenceDNA linker 12 cgtaggcgag aaagtgataa aaaaaaaaaa aaaaaaaaaa aaaaacc 47 1310 DNA Artificial Sequence DNA linker 13 tagcggatgc 10 14 19 DNAArtificial Sequence DNA linker 14 tttttttttt agcggatgc 19 15 39 DNAArtificial Sequence DNA linker 15 taatacgact cactataggg acaattactatttacaatt 39

What is claimed is:
 1. A method for generating a DNA-protein fusion,said method comprising: (a) covalently bonding a nucleic acidreverse-transcription primer to an RNA, said reverse-transcriptionprimer being bound to a peptide acceptor; (b) translating said RNA toproduce a protein product, said protein product being covalently boundto said reverse-transcription primer; and (c) reverse transcribing saidRNA to produce a DNA-protein fusion.
 2. A method for generating aDNA-protein fusion, said method comprising: (a) generating anRNA-protein fusion; (b) hybridizing a nucleic acid reverse-transcriptionprimer to said fusion; (c) covalently bonding said primer to saidfusion; and (d) reverse transcribing the RNA of said RNA-protein fusionto produce a DNA-protein fusion.
 3. The method of claim 1 or 2, saidmethod further comprising treating the product of step (c) to removesaid RNA.
 4. The method of claim 3, wherein said treating comprisescontacting the product of step (c) with RNase H under conditionssufficient to digest said RNA.
 5. The method of claim 1 or 2, whereinsaid nucleic acid reverse-transcription primer is a DNA primer.
 6. Themethod of claim 1 or 2, wherein said translating step is carried out invitro.
 7. The method of claim 1 or 2, wherein said peptide acceptor ispuromycin.
 8. The method of claim 1, wherein said nucleic acidreverse-transcription primer has a hairpin structure.
 9. The method ofclaim 1 or 2, wherein said nucleic acid reverse-transcription primerfurther comprises a photocrosslinking agent.
 10. The method of claim 9,wherein said photocrosslinking agent is psoralen.
 11. The method ofclaim 9, wherein said crosslinked to an oligonucleotide which is boundto said peptide acceptor.
 12. The method of claim 9, wherein saidnucleic acid reverse-transcription primer is hybridized to said RNAmolecule and said linking step is carried out by photocrosslinking. 13.A method for generating a DNA-protein fusion, said method comprising:(a) providing an RNA molecule covalently bonded to a peptide acceptor;(b) covalently bonding a nucleic acid reverse-transcription primer tothe molecule of step (a); (c) translating said RNA molecule to produce aprotein, and (d) reverse transcribing said RNA molecule to produce aDNA-protein fusion.
 14. The method of claim 2 or 13, wherein the proteinof said DNA-protein fusion is covalently bonded to the RNA through anon-RNA linker and said nucleic acid reverse-transcription primer iscovalently bonded to said linker.